CA1047422A

CA1047422A - Process of manufacturing enzyme preparation rich in lipase

Info

Publication number: CA1047422A
Application number: CA219,898A
Authority: CA
Inventors: Hans Betzing; Dac Lekim
Original assignee: A Natterman und Cie GmbH
Current assignee: A Natterman und Cie GmbH
Priority date: 1974-02-21
Filing date: 1975-02-12
Publication date: 1979-01-30
Also published as: CH623848A5; DK136728C; SE7501873L; DK68275A; DK136728B; AT338415B; NL7501945A; ATA136075A; JPS50123872A; JPS5328514B2; ES434840A1; US3925158A; SE424086B; FR2262045B1; PH11983A; GB1454983A; FR2262045A1

Abstract

ABSTRACT OF THE DISCLOSURE
Enzyme preparation rich in lipase is obtained by treating comminuted pancreas tissue with a mixture of about 9 parts by volume of chloroform and one part butanol. The partly degreased tissue is then left standing at 0-4°C for 24 to 96 hours. Subsequently, it is degreased through treat-ment with acetone and is then dehydrated, which is followed by an extraction with a 5% aqueous ethanol solution. The extract is then mixed with acetone, the obtained precipitate is isolated and dried.

Description

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` The present invention relates to enzyme preparations : and, more particularly, to the preparation of pancreas enzyme rich in lipase. Lipase is identified by enzyme number 3.1.1.3.
in "Comprehensive ~iochemistry", Vol. 13, 1973, 3rd Edition, Florkin et al, Elsevier Pub. Co., New York.
It has been known for a long time that lipase-containing pancreas preparations may be used for the fermenta-tive cleavage of fats. Numerous publications describe the j~..,~
'- concentration of the lipase in such enzyme preparations. Among ~ 10 other possibilities, such preparations are used in the treatment ., .
of indigestion and the effects thereof, whose causes are based ' above all on an insufficient excretion of the pancreas or on .,; .
an insufficient production of the enzymes in question.
The enzyme preparations available commercially at the present time ordinarily exhibit lipase activities of 40 to 60 Willstatter units (WE~/g; a WE is an amount of enzyme . , , that splits 24% oil in 2.5 g olive oil in one hour under determined conditions. However, the activity values of such preparations are very frequently unsatisfactory for therapeutic use in man, according to medical experience, since the deficiency of lipase cannot be compensated with the customary doses of the preparations.
British Patent 1,328,202 discloses a process of preparing a pancreatin preparation possessing an activity higher than 3 x NF, in which connection NF indicates the minimal pancreastin activity of such preparations in relation to starch and casein, published by the National Formulary, XIIth Edition, of the American Pharmaceutical Association, Washington, D.C., U.S.A. According to this process, the comminuted pancreas glands are subjected first of all to an autolysis in an aqueous alkaline medium at a temperature of 20 - 30C, prior to de-hydration and removal of fat with propyl or butyl alcohol.
~owever, the enzyme preparation obtained from the autolysed tissue material after such degreasing still exhibits a rela-~ A~ - 2 - ~ `
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tively low lipase activity.
Now, it has been unexpectedly found that a strong increase in activity especially of the lipase is obtained in the extract material when the autolysis is performed at low .:
temperatures after a partial degreasing and dehydration of i the comminuted pancrease-gland material and subsequent extrac-tion of the material. This could not have been expected, since in the process of the sritish Patent 1,328,202 it was possible to increase substantially the amylase activity through the stage of autolysis, but it was not possible to have such .. . .
an increase in the lipase activity.
According to the present invention, there is provided an improvement in the process of preparing an enzyme prepar-ation having a high content of lipase (enzyme number 3.1.1.3-) by comminution of pancreas tissue, autolysis, removal of fat and aqueous extraction of the tissue, and recovery of the enzyme from the aqueous extract, the improvement wherein the comminuted pancreas tissue is treated with a mixture of about 9 parts by volume of chloroform and 1 part butanol, the :,. . .
partly degreased tissue is then left standing at 0-4C for ", 24 to 96 hours to effect the autolysis.
In a preferred embodiment, after autolysis, the tissue is degreased through treatment with acetone and is then de-, hydrated, which is followed by an extraction with a 5% aqueous - ethanol solution. The extract is then mixed with acetone, the obtained precipitate is isolated and dried.
Generally, the process of the invention is performed in such a manner that immediately after thawing the deep-cooled pancreas tissue is comminuted in a meat grinder or mixer together with a 9:1 mixture of chloroform and butanol. After the organic solvent is removed, the tissue is again treated with the same solvent mixture while being stirred and the ~' ' , -- ~474ZZ
solvent is again decanted, whereafter the still-moist residue is left standing at least for 24 hours, preferably 48 hours, in a cooler at 0-4C, for the purpose of performing the autolysis.
;`~ After the autolysis, the already largely degreased tissue material is stirred thoroughly twice with acetone and the solvent is separated in each case. For isolating the enzyme material which is rich in lipase, the dry tissue powder is repeatedly stirred with a mixture of water and ethanol.
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(volume ratio 95 : 5), the solutions separated from the tissue are combined and the precipitate is separated through addition of acetone and subsequent centrifugation of the so formed f. .
suspension. The precipitate is then treated with acetone and, . .; , subsequently, dried in vacuo. The obtained preparation is a fine gray-white powder possessing an unusually high lipase : , .
activity.
Since in the obtained water-alcohol extracts the :.~
enzyme already is present in a high degree of purity, such 5% alcohol extracts may also be employed in accordance with an :.,;
~`, embodiment of the process directly for the production of the lipase preparation, by subjecting the combined extracts to , .
, ~ freeze drying in which the enzyme preparation rich in lipase ~! .
~`~ is obtained in the form of dry, gray-white powder. The advantag~
~, 15 of this modification of the process is based on the fact that r~ .
recovery is simplified, i.e. the precipitation of the enzyme ,;; preparation with acetone and the isolation of the obtained , ,,~ .
~- precipitate through filtering or centrifugation are eliminated. -In addition to a great economy of time, this procedure especially results also in the saving of large amounts of acetone, whose recovery is rendered difficult because of the water content.
The activity of the lipase preparation thus obtained corresponds to that of the enzyme obtained through the precipitation with acetone.
The following working examples are offered illustratively:

1~474Z2 EXAMPLE
After thawing, 5.0 kg deep-cooled hog pancreas are ; freed of adhering fat, coarsely comminutedand homogenized with 5 1 of a mixture of chloroform-butanol (9 : 1) in a mixer or meat grinder. The formed organ paste is stirred for 30 minutes at room temperature, the chloroform-containing liquid phase . .
: is decanted off and the residue is then still stirred twice, , with 5 1 chloroform-butanol ~9 : l) in each case, for 30 minutes .: . .
at room temperature, and the solvent is decanted off in each case. The residue which is still moist is then left alone for .. , ~ .
48 hours at 0 - 4C. Subsequently, the organ paste, which is ~- thus largely degreased, is stirred thoroughly twice, with 2.5 1 acetone in each case, for 15 minutes, and the supernatant solution is decanted off in each case.
For extracting the ferments rich in lipase, the resultant entirely degreased paste is then stirred twice for :
30 minutes, with 2 1 of a mixture of water and ethanol (95 : 5) in each case, and the solution is filtered off subsequently.
~he separate water-ethanol extracts are combined and stirred wlth 5 1 acetone for 15 minutes at room temperature, for the ~ purpose of precipitating the pancreas ferments. After - - centrifugation to recover the precipitate, it is wahsed three : times with 1.6 1 acetone in each case. Then the washed pancreas -ferment precipitate is dried in vacuo and comminuted.
Yield: 450 g (9% in regard to the moist organ) of a yellowish powder having a lipase activity of 270 WE/g.

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Two kg of freeze-dried hog pancreas are extracted ~ three times for 30 minutes, with 5 1 chloroform-butanol (9 : 1) : in each case, while being stirred, and the solvent is decanted . .
off in each case. The moist residue is then left alone for 60 hours at 0 - 4C. In order to complete the degreasing, the material is then stirred twice for 15 minutes, with 2.5 1 acetone in each case, and filtered in each case from the solvent.
r~"' . For extracting the pancreas ferments, the degreased organ residue is treated twice for 30 minutes with 2 1 of a ~, - - - , .
mixture of water and ethanol (95 : 5) in each case, while tirred, the solution being filtered off in each case from the ~- residue. The combined water-ethanol extracts are stirred t'~ .
; with 5 1 acetone for 15 minutes at room temperature, and the i 15 resultant precipitated ferments rich in lipase are separated . .
by centrifugation. The precipitate is then washed three times ~;q with 1.6 1 acetone in each case, dried in vacuo and comminuted.
~ EXAMPLE 3 ;~ Three kg of deep-cooled hog pancreas is comminuted - 20 very finely in a meat grinder and homogenized in a mixer with 3.0 liter of a mixture of chloroform-butanol at 9:1 v/v. The corresponding organ paste is stirred for 30 minutes at room temperature, the chloroform-containing liquid phase is drained off and the residue is further stirred twice for 30 minutes at room temperature, in each case with 3.0 1 chloroform-butanol at 9:l v/v and the solvent is separated in each case. The i ~ . ~
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still-moist residue is then left alone for 48 hours at 4C and ..;
the largely degreased organ is subsequently again stirred . .. .
- twice for 15 minutes, in each case with 1.5 1 acetone and the supernatant solution is decanted off in each case.
, .
~ 5 In order to extract the pancreas ferments that are . .
rich in lipase, the entirely degreased material is stirred twice for 30 minutes, in each case with 1.5 liter of a mixture of water and ethanol at 95:5 v/v and the solution is then filtered through a metal screen. The combined water-ethanol extracts are freeze-dried to remove the solvent, using the following conditions: Thickness of liquid layer about 1 - 2 cm, initial temperature - 20C, final pressure 10-4 torr, final -temperature 20C. The dried porous material is comminuted and screened.
Yield: 365 g (12% in regard to the moist organ) of a light yellow powder having a lipase activity of about 250 Willstatter units/g, corresponding to 110 000 FIP units/g.
, . ~ .

,' A comparison of the pancreatin produced in accordance with the examples of the British Patent with a product prepared in accordance with the process of the present invention shows that, through the gentle autolysis of the present invention, without an addition of bases or acids, namely by merely standing aione for at least 24 hours at 0 - 4C and with the modified refining-concentration method, one obtains a pancreatin prepara-tion that possesses a substantially higher lipase activity.

lQ4742Z
Pancreatin preparation Lipase activity in WE/g produced in accordance with Example 1 of the British Patent 1,328,202 95 , 5 produced in accordance with the process of the present invention 240 , . The decisive role of the temperature at which the autolysis takes place follows clearly from the following table:
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T A B L E

~ , .
.~- 10 Activity "
. Duration of autolysis Autolysis at Autolysis at in hours room temperature 0 - 4C
.: 0 100% 100%
, 24 97% 110%
: 48 52% 150%
1596 _ 140%

The following Table 2 shows the lipase activities of two commercial pancreatin preparations in comparison with the corresponding activities of the products rich in lipase, as obtained in accordance with the process of the present application, the lipase activities being determined by the method of Willstatter and Lazo-Wasem in Willstatter and Wilson units, respectively.

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, '`'`''' ~' T A B L E 2 .. '': _ .
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''.~ ' Lipase activity Lipase activity '. Product in accordance in accordance .: with Willstatter with Lazo-Wasem ;:
'. _. . (in Wilson units) !' Commercial 4-NF-product 45 WE/g 2060 units/g ~' - Commercial 5/6-NF-product 56 WE/g 24S0 units/g v''~ 5 Product of the'invention 239.6 WE/g 8000 units/g ..... _ . . :, ., , ~ In these experiments the lipase activity was deter-'-, mined in accordance with Willstatter (Hoppe-Seylers 125 (1923) " 193) modified according to Vogel and Laeverenz (Hoppe-Seylers ':~
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' 234 (1935) 176), by foaming 2 - 3 mg of the pancreas prepara- :~
tion in 5 ml water and 2 ml NH3-NH4Cl-buffer of pH 9.2, then' -~
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:s mixing in each case with 2 ml of (2.4%) egg albumen solution,' (1.6%) CaC12 solution and (1.6%) sodium oleate solution and finally adding to 2.5 g olive oil. The preparation was ' vigorously shaken for a few seconds and then mixed thoroughly ~ . 15 in a magnetic stirrer for 60 minutes at 30C. Then the cleavage ' -~ -was interrupted by adding 100 ml ethanol and, after the addition ' of 20 ml ether and 12 drops of 1% alcoholic thymolphthalein 'solution, the material was titrated to blue color with 0.5 n '' alcoholic KOH. From the consumption of lye thus determined, ' 20 one substracts the consumption of the blank test that is started -simultaneously with the main test, with the difference that the . olive oil is added only after the addition of alcohol and ether, .

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i.e., shortly before the titration. From the percentage of cleavage thus obtained, the corresponding lipase units are ~- calculated by means of an empirically determined calibration ~ .
curve. The Willstatter units (WE)/g are obtained by multiplying the lipase units with the factor 1000 ` mg weight-in amount When the lipase is determined in accordance with ~ E.A. Lazo-Wasem ~J. of Pharm. Sciences 50 (1961) 999], the .... ~ .
olive oil is treated with the enzyme preparation for 30 minutes at 37C and at a pH value of 7.8 while beef gall is added.
v~ 10 After the acidification the split-off fatty acids are extracted with benzene and titrated with phenolphthalein as indicator.
~-i It turns out that the lipase activities of the enzyme preparations of the invention are situated far above the values ., .
of the commercial pancreatin products in accordance with both methods of determination. Stability tests have shown that the high lipase activities on which the novel enzyme preparations -of the present invention are based do not show practically any change when stored for several months at room temperature.
It is particularly important that the novel pancreatin preparations of the present invention are not only strongly ~; enriched in regard to lipolytic enzymes but, even if to a .
lesser extent, also exhibit an increase in protease and amylase activities in relation to the customary commercial pancreatins.
In the following Table 3, the protease, amylase and esterase ',J 25 activities of the pancreatins of the invention are compared with a commercial 5/6-NF-product.

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It is clear that the process of the invention ' strongly enriches especially the lipase activity.
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~ It will be obvious to those skilled in the art that , ~.: various changes may be made without departing from the scope ~ ' .
~ 5of the invention and the invention is not to be considered ... .
"rlimited to what is shown and described in the specification.
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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. In a process of preparing an enzyme preparation rich in lipase (enzyme number 3.1.1.3.) by comminution of pancreas tissue, autolysis, removal of fat and aqueous extraction of the tissue, and recovery of the enzyme from the aqueous extract, the improvement wherein:
the comminuted pancreas tissue is first partially degreased with a mixture of about 9 parts by volume of chloro-form and 1 part by volume butanol, then the partially degreased tissue material is left standing for 24-96 hours at 0-4° to effect autolysis, and then after said aqueous extraction, said recovery is carried out.

2. A process in accordance with claim 1, wherein after said autolysis the tissue is degreased with acetone and de-hydrated, whereafter extraction is performed with 5% aqueous ethanol solution and the dry enzyme preparation is recovered from the extract.

3. A process as in claim 1, wherein the partly degreased tissue is left standing for 48 hours at 0-4°C.

4. A process in accordance with claim 1, wherein following said aqueous extraction, said recovery is affected by freeze drying the aqueous extract.